Mammalian cell surface antigens; related reagents

ABSTRACT

Purified genes encoding a T cell surface antigen from a mammal, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding this antigen are provided. Methods of using said reagents and diagnostic kits are also provided.

This filing is a continuation of U.S. patent application Ser. No.13/963,332, filed Aug. 9, 2013; which is a continuation of U.S. patentapplication Ser. No. 13/742,189, filed Jan. 15, 2013, now abandoned;which is a continuation of U.S. patent application Ser. No. 13/331,879,filed Dec. 20, 2011, now abandoned; which is a Continuation of U.S.patent application Ser. No. 11/351,617, filed Feb. 10, 2006; nowabandoned, which is a Divisional of U.S. patent application Ser. No.09/545,998, filed Apr. 10, 2000, now U.S. Pat. No. 7,025,962; which is aDivisional of U.S. patent application Ser. No. 08/911,423, filed Aug.14, 1997, now U.S. Pat. No. 6,111,090; which claims benefit from U.S.Provisional Patent Application Nos. 60/027,901, filed Oct. 7, 1996; and60/023,419, filed Aug. 16, 1996, each of which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention generally pertains to molecules that controlactivation and expansion of mammalian cells, especially mammalian immunesystem cells. The invention provides purified genes, proteins,antibodies, and related reagents useful, for example, to regulateactivation, development, differentiation, and function of various celltypes, including hematopoietic cells. In particular, the inventionprovides mammalian 312C2 genes, gene products, compositions, and methodsfor using these.

BACKGROUND OF THE INVENTION

The activation of resting T cells is critical to most immune responsesand allows these cells to exert their regulatory or effectorcapabilities. See Paul (ed; 1993) Fundamental Immunology 3d ed., RavenPress, N.Y. Increased adhesion between T cells and antigen presentingcells (APC) or other forms of primary stimuli, e.g., immobilizedmonoclonal antibodies (mAb), can potentiate the T-cell receptor signals.T-cell activation and T cell expansion depends upon engagement of theT-cell receptor (TCR) and co-stimulatory signals provided by accessorycells. See, e.g., Jenkins and Johnson (1993) Curr. Opin. Immunol.5:361-367; Bierer and Hahn (1993) Semin. Immunol. 5:249-261; June, etal. (1990) Immunol. Today 11:211-216; and Jenkins (1994) Immunity1:443-446. A major, and well-studied, co-stimulatory interaction for Tcells involves either CD28 or CTLA-4 on T cells with either B7 or B70(Jenkins (1994) Immunity 1:443-446). Recent studies on CD28 deficientmice (Shahinian, et al. (1993) Science 261:609-612; Green, et al. (1994)Immunity 1:501-508) and CTLA-4 immunoglobulin expressing transgenic mice(Ronchese, et al. (1994) J. Exp. Med. 179:809-817) have revealeddeficiencies in some T-cell responses though these mice have normalprimary immune responses and normal CTL responses to lymphocyticchoriomeningitis virus and vesicular stomatitis virus. As a result, boththese studies conclude that other co-stimulatory molecules must besupporting T-cell function. However, identification of these moleculeswhich mediate distinct costimulatory signals has been difficult.

The inability to modulate activation signals prevents control ofinappropriate developmental or physiological responses in the immunesystem. The present invention provides at least one alternativecostimulatory molecule, agonists and antagonists of which will be usefulin modulating a plethora of immune responses.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the discovery of a familyof proteins which appear to act as a costimulator of T cell activation.In particular, the invention provides mammalian, e.g., rodent andprimate, genes designated m312C2 and h312C2, respectively, which areexpressed in the thymus, and are induced on T cells and spleen cellsfollowing activation. Engagement of 312C2 appears stimulateproliferation of T cell clones, antigen-specific proliferation andcytokine production by T cells, and appears to potentiate T cellexpansion or apoptosis. The mouse and human embodiments are described ingreater detail, but the invention encompasses related mammalian genes,proteins, antibodies, and uses thereof. Functional equivalentsexhibiting significant sequence homology are available from othermammalian and non-mammalian species. Moreover, the ligand of 312C2 canfunction as its binding partner to stimulate other cells expressing theantigen.

The present invention provides a substantially pure or recombinant 312C2protein or peptide fragment thereof. The protein or polypeptide isexpressed, e.g., activated T cells or specifically binds to antibodiesgenerated against SEQ ID NO: 2 or 4. Some embodiments involve a proteinor peptide selected from a protein or peptide from a warm blooded animalselected from the group of birds and mammals, including a rodent orprimate. The groups further consist of a protein or peptide whichcomprises at least one polypeptide segment of SEQ ID NO: 2 or 4; is notglycosylated; is in a buffered solution; is attached to a solidsubstrate; exhibits a plurality of epitopes from SEQ ID NO: 2 or 4; issynthetically labeled; is conjugated to a chemical moiety; is a 5-foldor less substitution from a natural sequence; or is a deletion orinsertion variant from a natural sequence. The protein or peptide cancomprise a sequence from the extracellular or the intracellular portionof a 312C2; or be a fusion protein.

The invention also provides a recombinant nucleic acid comprisingsequence at least about 70% identity over a stretch of at least about 30nucleotides to a 312C2 nucleic acid sequence of SEQ ID NO: 1, 3 or 5,useful, e.g., as a probe or PCR primer for a related gene. Anotherembodiment further encodes a polypeptide sharing a plurality of specificepitopes and comprising at least about 60% identity over a stretch of atleast about 20 amino acids to a 312C2 sequence of SEQ ID NO: 2 or 4.

Another embodiment is a sterile composition comprising a 312C2 proteinand a pharmaceutically acceptable carrier. Other compositions maycombine said entities with an agonist or antagonist of other T cellsignaling molecules, e.g., signaling entities through the T cellreceptor, CD40, CD40 ligand, CTLA-8, CD28, B7, B70, BAS-1, SLAM, etc.

The invention also embraces an antibody which specifically binds a 312C2protein or peptide, e.g., wherein the 312C2 is a mammalian protein,including a mouse; the antibody is raised against a purified 312C2peptide sequence of SEQ ID NO: 2 or 4; the antibody is a monoclonalantibody; the antibody is detectably labeled; the antibody is attachedto a solid substrate; or the antibody is in a sterile or bufferedcomposition. The antibodies also make available a method of purifying a312C2 protein or peptide from other materials in a mixture comprisingcontacting the mixture to an anti-312C2 antibody, and separating bound312C2 from other materials.

Another aspect of the invention is an isolated or recombinant nucleicacid capable of encoding a 312C2 protein or peptide, including a nucleicacid which encodes a mature polypeptide sequence of SEQ ID NO: 2 or 4;which includes a sequence of SEQ ID NO: 1, 3 or 5; which encodes asequence from an extracellular domain of a natural 312C2; which encodesa sequence from an intracellular domain of a natural 312C2; which isdetectably labeled; which is attached to a solid substrate; or is in asterile composition. Such nucleic acid embodiments also include anexpression or replicating vector.

Also provided is a method of expressing a 312C2 peptide by expressing anucleic acid encoding a 312C2 polypeptide. The invention also provides acell, tissue, organ, or organism comprising a nucleic acid encoding a312C2 peptide.

The invention also provides a kit containing a substantially pure 312C2or fragment; an antibody or receptor which specifically binds a 312C2;or a nucleic acid, or its complement, encoding a 312C2 or peptide. Thiskit also provides methods for detecting in a sample the presence of anucleic acid, protein, or antibody, comprising testing said sample withsuch a kit.

The invention also supplies methods of modulating the physiology of acell comprising contacting said cell with a substantially pure 312C2 ora fragment thereof; or with an antibody or ligand which specificallybinds a 312C2; or with a nucleic acid encoding a 312C2 or a peptidefragment thereof. Certain preferred embodiments include a method wherethe cell is a T cell and the modulating of physiology is activation ofthe T cell or apoptosis of the T cell; or where the cell is in a tissueand/or in an organism.

The invention further provides a method of treating a patient having anabnormal immune response by administering an effective dose of anantibody or binding partner specific for 312C2; a 312C2 protein orpolypeptide; or a nucleic acid encoding a 312C2 peptide. The abnormalimmune response is characterized by a T cell immune deficiency; chronicinflammation; or tissue rejection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The terms “nucleic acid” “probe”, or “primer” include reference to adeoxyribonucleotide, ribonucleotide, or mixed polymer in either single-or double-stranded form, and unless otherwise limited, encompasses knownanalogs of natural polynucleotides that hybridize to nucleic acids inmanner similar to naturally occurring polynucleotides. Unless otherwiseindicated, a particular nucleic acid sequence includes the perfectcomplementary sequence thereof. Eukaryotic nucleic acids are nucleicacids from eukaryotic cells, preferably cells of multicellulareukaryotes.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. The terms defined below are morefully defined by reference to the Specification as a whole.

The term “recombinant” when used with reference to a cell, or nucleicacid, or vector, includes reference to a cell, or nucleic acid, orvector, that has been modified by the introduction of a heterologousnucleic acid or the alteration of a native nucleic acid to a form notnative to that cell, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

The term “subsequence” in the context of a referenced nucleic acidsequence includes reference to a contiguous sequence from the nucleicacid having fewer nucleotides in length than the referenced nucleicacid. In the context of a referenced protein, polypeptide, or peptidesequence (collectively, “protein”), “subsequence” refers to a contiguoussequence from the referenced protein having fewer amino acids than thereferenced protein. The terminus of such subsequences include allcombinations consistent with a defined length.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations”, whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describesequivalents which encompass every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine) canbe modified to yield a functionally identical molecule. Accordingly,each silent variation of a nucleic acid which encodes a polypeptide isimplicit in each described sequence. Substitutions with functionallyequivalent unusual nucleotides or analogs are intended, e.g., inositol,etc.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. The following six groups each containamino acids that are conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See also,        Creighton (1984) Proteins W.H. Freeman and Company.

By “contiguous amino acids from” in the context of a specified number ofamino acid residues from a specified sequence, is meant a sequence ofamino acids of the specified number from within the specified referencesequence which has the identical order of amino acids each of which isdirectly adjacent to the same amino acids as in the reference sequence.

The term “polypeptide” as used herein includes a significant fragment orsegment, and encompasses a stretch of amino acid residues of at leastabout 8 amino acids, generally at least about 12 amino acids, typicallyat least about 16 amino acids, preferably at least about 20 amino acids,and, in particularly preferred embodiments, at least about 30 or moreamino acids, e.g., 40, 50, 60, 70, 80, 100, etc. The ends may be arevirtually all combinations consistent with length.

The term “plurality of non-overlapping fragments” encompasses a seriesof polypeptide fragments or segments. A plurality includes 2, 3, 4, 5,etc., polypeptide fragments.

The terms “biologically pure” or “isolated” refer to material which issubstantially or essentially free from components which normallyaccompany or interact with it as found in its naturally occurringenvironment. The isolated material optionally comprises material notfound with the material in its natural environment.

The phrase “encodes a protein” in the context of nucleic acids includesthose nucleic acids encoding naturally occurring proteins or derivativesof natural proteins, but which are deliberately modified or engineeredto no longer hybridize to a natural gene encoding the protein of naturalorigin under the stated conditions.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison may be conducted by the local homology algorithm of Smithand Waterman (1981) Adv. Appl. Math. 2:482; by the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-445; bythe search for similarity method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. USA 85:2444; by computerized implementations of thesealgorithms (including, but not limited to CLUSTAL in the PC/Gene programby Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), 575 Science Dr., Madison, Wis., USA); the CLUSTAL programis well described by Higgins and Sharp (1988) Gene 73:237-244 andHiggins and Sharp (1989) CABIOS 5:151-153; Corpet, et al. (1988) NucleicAcids Research 16:10881-90; Huang, et al. (1992) Computer Applicationsin the Biosciences 8:155-65, and Pearson, et al. (1994) Methods inMolecular Biology 24:307-31. Alignment is also often performed byinspection and manual alignment.

The terms “identical” or “sequence identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g. chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well-known to those of skill in the art. Typically thisinvolves scoring a conservative substitution as a partial rather than afull mismatch, thereby increasing the percentage sequence identity.Thus, for example, where an identical amino acid is given a score of 1and a non-conservative substitution is given a score of zero, aconservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller (1988) Computer Applic. Biol. Sci.4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

The terms “substantial identity” or “similarity” of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 60% sequence identity, preferably at least 80%, more preferably atleast 90%, and most preferably at least 95%, compared to a referencesequence using, e.g., the programs described above (preferably BLAST)using standard parameters. One indication that two nucleic acidsequences are substantially identical is that the polypeptide which thefirst nucleic acid encodes is immunologically cross reactive with thepolypeptide encoded by the second nucleic acid.

Another indication that two nucleic acid sequences have substantialidentity is that the two molecules hybridize to each other under“moderate stringency hybridization conditions” (or “moderateconditions”) or better. Exemplary “moderate stringency hybridizationconditions” include a hybridization in a buffer of 40% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positivehybridization is at least twice background. Those of ordinary skill willreadily recognize that alternative hybridization and wash conditions canbe utilized to provide conditions of similar or higher stringency.Nucleic acids which do not hybridize to each other under moderatestringency hybridization conditions are still substantially identical ifthe polypeptides which they encode are substantially identical. Thisoccurs, e.g., when a copy of a nucleic acid is created, e.g., using themaximum codon degeneracy permitted by the genetic code.

The terms “substantial identity” or “similarity” in the context of apeptide indicates that a peptide comprises a sequence with at least 60%sequence identity to a reference sequence, usually at least 70%,preferably 80%, more preferably 85%, most preferably at least 90% or 95%sequence identity to the reference sequence over a specified comparisonwindow. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443.An indication that two peptide sequences are substantially identical isthat one peptide is immunologically reactive with antibodies raisedagainst the second peptide. Thus, a peptide is substantially identicalto a second peptide, for example, where the two peptides differ only bya conservative substitution. Generally, similarity is determined using acomparison window having a length of any number from 20 contiguouspositions at various positions in the respective molecules to the numberof residues in the full-length core region sequence, where thecomparison window is within the core sequence.

The terms “oligonucleotide” or “polynucleotide” probes include referenceto both double stranded and single stranded DNA or RNA. The terms alsorefer to synthetically or recombinantly derived sequences essentiallyfree of non-nucleic acid contamination.

As used herein, “contact” or “contacting” means to place in directphysical association, e.g., mixing of solutions.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains, or is being tested for presence of, 312C2 protein,or another of described composition, e.g., nucleic acid or protein. Suchsamples include, but are not limited to, sputum, amniotic fluid, blood,blood cells, e.g., white cells, or tissue, e.g., spleen, thymus, bonemarrow, or lymph node. Biological samples may also include sections oftissues such as frozen sections taken for histological purposes.Examples of biological samples include a cell sample from nervous,muscular, glandular or epithelial tissue or from the immune system(e.g., T cells). A biological sample is typically obtained from aeukaryotic organism, preferably a multicellular eukaryotes such asinsect, protozoa, birds, fish, reptiles, and preferably a mammal such asrat, mice, cow, dog, guinea pig, pig, goat, or rabbit, and mostpreferably a primate such as macaques, chimpanzees, or humans.

An “expression vector” is a nucleic acid construct, typically generatedrecombinantly or synthetically, with a series of specified nucleic acidelements which permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed, and a promoter.

The phrase “functional effects” in the context of assays for testingcompounds affecting the 312C2 includes the determination of anyparameter that is indirectly or directly under the influence of the312C2. It includes changes such as increases or decreases oftranscription or second messenger or lymphokine release.

By “selectively hybridizing” or “selective hybridization” or“selectively hybridizes” is meant hybridization, under stringenthybridization conditions, of a nucleic acid sequence to a specifiednucleic acid target sequence to a detectably greater degree than itshybridization to non-target nucleic acid sequences and/or to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences typically have at least 80% sequence identity,usually 90% sequence identity, preferably 95% identity, more preferably98% identity, and most preferably 100% sequence identity (i.e.,complementary) with each other over lengths which typically start fromabout 10 nucleotides, e.g., 13, 17, 20, 23, 26, 29, 32, etc. “Percentageof sequence identity” is determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) as compared to the reference sequence (whichdoes not comprise additions or deletions) for optimal alignment of thetwo sequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity.

The terms “stringent conditions” or “stringent hybridization conditions”refer to conditions under which a probe will hybridize to its targetsequence, to a detectably greater degree than other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures. Generally, stringent conditions are selected to be about5° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of30% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 2×SSC at 50° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C.

“Stringent hybridization conditions” or “stringent conditions” in thecontext of nucleic acid hybridization assay formats are sequencedependent, and are different under different environmental parameters.An extensive guide to the hybridization of nucleic acids is found e.g.,in Tijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.

By “hybridization complex” is meant a duplex nucleic acid sequenceformed by selective hybridization of two single-stranded nucleic acidsequences with each other.

By “host cell” is meant a cell which is manipulated to contain and, incertain instances, express a molecule, usually a nucleic acid. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells.

The term “antibody” also includes antigen binding forms of antibodies(e.g., Fab, F(ab)₂). The term “antibody” refers to a polypeptidesubstantially encoded by an immunoglobulin gene or immunoglobulin genes,or fragments thereof which specifically bind and recognize an analyte(antigen). Antibodies exist, e.g., as intact immunoglobulins or as anumber of well characterized fragments e.g., produced by digestion withvarious peptidases. Thus, for example, pepsin digests an antibody belowthe disulfide linkages in the hinge region to produce F(ab)′₂, a dimerof Fab which itself is a light chain joined to V_(H)-C_(H)1 by adisulfide bond. The F(ab)′₂ may be reduced under mild conditions tobreak the disulfide linkage in the hinge region, thereby converting theF(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially anFab with part of the hinge region, see, e.g., Paul (ed.) (1993)Fundamental Immunology, 3rd ed., Raven Press, N.Y. While variousantibody fragments are defined in terms of the digestion of an intactantibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term antibody, as used herein, is oftenfunctionally equivalent to antibody fragments such as single chain Fv,chimeric antibodies (i.e., comprising constant and variable regions fromdifferent species), humanized antibodies (i.e., comprising acomplementarity determining region (CDR) from a non-human source) andheteroconjugate antibodies (e.g., bispecific antibodies).

By “immunologically reactive conditions” is meant conditions which allowan antibody, generated to a particular epitope, to bind to that epitopeto a detectably greater degree than the antibody binds to substantiallyall other epitopes. Immunologically reactive conditions are dependentupon the format of the antibody binding reaction and typically are thoseutilized in immunoassay protocols. See Harlow and Lane (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions.

By “antibody reactive to a protein” is meant the protein is“specifically immunoreactive with an antibody.”

The phrase “specifically immunoreactive with an antibody”, or“specifically binds to an antibody” when referring to a protein orpeptide, refers to a binding reaction between an antibody and a proteinhaving an epitope recognized by the antigen binding site of theantibody. This binding reaction is determinative of the presence of aprotein having the recognized epitope amongst the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aprotein having the recognized epitope and bind, if at all, to adetectably lesser degree to other proteins lacking the epitope which arepresent in the sample.

Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.For example, antibodies raised to the 312C2 of SEQ ID NO: 2 or 4 can beselected from to obtain antibodies specifically immunoreactive with thatparticular protein and not with other proteins. The proteins used asimmunogens can be in native conformation or denatured, e.g., so as toprovide a linear epitope. Preferably, antibody preparations whichspecifically recognize multiple epitopes will be used.

A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with a protein. See Harlow andLane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

By “antigen” is meant a substance to which an antibody can be generatedand/or to which the antibody is specifically immunoreactive with. Anantibody immunologically reactive with a particular antigen can begenerated in vivo or by recombinant methods such as selection oflibraries of recombinant antibodies in phage or similar vectors. See,e.g., Huse et al. (1989) Science 246:1275-1281; and Ward, et al. (1989)Nature 341:544-546; and Vaughan et al. (1996) Nature Biotechnology,14:309-314.

By “transfected” is meant the introduction of a nucleic acid into aeukaryotic cell where the nucleic acid may be incorporated into thegenome of the cell (i.e., chromosome, plasmid, or mitochondrial DNA),converted into an autonomous replicon, or transiently expressed (e.g.,transfected mRNA). The transfection can be in vivo or ex vivo. “Ex vivo”means outside the body of the organism from which a cell or cells isobtained or from which a cell line is isolated. Ex vivo transfection ispreferably followed by re-infusion of the cells back into the organism.In contrast, by “in vivo” is meant within the body of the organism fromwhich the cell was obtained or from which a cell line is isolated.

The term “binding composition” refers to molecules that bind withspecificity to 312C2, e.g., in a cell adhesion pairing type fashion, oran antibody-antigen interaction. It also includes compounds, e.g.,proteins, which specifically associate with 312C2, including in anatural physiologically relevant protein-protein interaction, eithercovalent or non-covalent. The molecule may be a polymer, or chemicalreagent. A functional analog may be an antigen with structuralmodifications, or it may be a molecule which has a molecular shape whichinteracts with the appropriate binding determinants. The compounds mayserve as agonists or antagonists of the binding interaction, see, e.g.,Goodman, et al. (eds.) (1990) Goodman & Gilman's: The PharmacologicalBases of Therapeutics (8th ed.), Pergamon Press.

Substantially pure typically means that the protein is free from othercontaminating proteins, nucleic acids, or other biologicals with whichit is associated in the original source organism. Purity may be assayedby standard methods, typically by weight, and will ordinarily be atleast about 40% pure, generally at least about 50% pure, often at leastabout 60% pure, typically at least about 80% pure, preferably at leastabout 90% pure, and in most preferred embodiments, at least about 95%pure. Carriers or excipients will often be added, or aqueous buffers ororganic solvents used in certain situations.

II. General

The present invention provides amino acid sequences and DNA sequencesencoding various mammalian proteins which are antigens found in theearly stages of T cell activation, e.g., which can activate a T cell.Among these proteins are antigens which modulate, e.g., induce orprevent proliferation or differentiation of T cells, among otherphysiological effects. The full length antigens, and fragments, orantagonists will be useful in physiological modulation of cellsexpressing the antigen. The proteins will also be useful as antigens,e.g., immunogens, for raising antibodies to various epitopes on theprotein, both linear and conformational epitopes. The molecule may beuseful in defining or isolating functional T cell or NK cell subsets.

A cDNA encoding mouse 312C2 was isolated from an activated pro T-cellcDNA library, see Kelner, et al. (1994) Science 266:13995-1399. Themouse 312C2 cDNA contains a stretch of about 1073 bp in length andcontained one large open reading frame encoding a type I transmembraneprotein. Structural features include an N-terminal leader sequence ofabout 19 amino acids, an extracellular region of about 153 amino acids,a hydrophobic presumptive membrane spanning portion of about 25 aminoacids, and a presumptive cytoplasmic domain of about 50 amino acids. SeeSEQ ID NO: 2. A human cDNA was isolated using the mouse clone to probe ahuman anergic T cell library designated HY06. See SEQ ID NO: 3 and 4. Atransmembrane region may begin at about amino acid 155 and end at aboutamino acid 185 based on hydrophobicity analysis. The rodent and primatesequences can be aligned.

312C2 exhibits structural motifs characteristic of a member of the TNFreceptor family, with numerous cysteine repeats. Compare, e.g., with theCD40, OX40, TNF receptor, NGF receptor, and FASL receptor. Theintracellular portion 312C2 does not contain a death domain asdescribed, e.g., in Pan, et al. (1997) Science 277:815-818; andSheridan, et al. (1997) Science 277:818-821. Lack of the death domainmotif may indicate that 312C2 is likely to work in the control ofproliferation rather than apoptosis.

As used herein, the term “mouse 312C2” shall encompass, when used in aprotein context, a protein having amino acid sequence shown in SEQ IDNO: 2, or a significant fragment of such a protein, or another highlyhomologous protein derived from mouse. The term “human 312C2” shallencompass, when used in a protein context, a protein having amino acidsequence shown in SEQ ID NO: 4, or a significant fragment of such aprotein, or another highly homologous protein derived from human.

The natural antigens are capable of mediating various biochemicalresponses which lead to biological or physiological responses in targetcells. The embodiments characterized herein are from mouse and human,but other species and tissue specific variants exist. Additionalsequences for proteins in other mammalian species, e.g., primates androdents, should also be available. See below. The descriptions below aredirected, for exemplary purposes, to a mouse or human 312C2, but arelikewise applicable to related embodiments from other species.

III. Purified 312C2

The mouse 312C2 nucleic acid sequence is shown in SEQ ID NO: 1, and theamino acid sequence is shown in SEQ ID NO: 2, the human 312C2 nucleicacid sequence is shown in SEQ ID NO: 3, and the corresponding amino acidsequence is shown in SEQ ID NO: 4. A reverse translation of the human312C2 sequence is shown in SEQ ID NO: 5. These amino acid sequences,provided amino to carboxy, are important in providing sequenceinformation about the antigen allowing for distinguishing the proteinfrom other proteins and exemplifying numerous variants. Moreover, thepeptide sequences allow preparation of peptides to generate antibodiesto recognize such segments, and nucleotide sequences allow preparationof oligonucleotide probes, both of which are strategies for detection orisolation, e.g., cloning, of genes encoding such sequences.

The mouse 312C2 nucleotide and predicted amino-acid sequence,particularly the predicted leader sequence runs from about Met1 throughGly19, though natural boundaries may be different, also depending uponcell type. A polyadenylation signal occurs at nucleotide position 1010.The poly A tail begins at position 1034. See SEQ ID NO: 1 and 2. Thetransmembrane domain is predicted to encompass amino acids beginning atabout 154 through about 179.

In human the putative leader sequence runs from about Met1 through aboutLeu18. The transmembrane domain is predicted to begin at out amino acid153 through about 182. Again the natural boundaries may vary.

Antibodies to these proteins typically bind to a 312C2 with highaffinity, e.g., at least about 100 nM, usually better than about 30 nM,preferably better than about 10 nM, and more preferably at better thanabout 3 nM. Homologous proteins would be found in mammalian speciesother than mouse, e.g., primates or rodents. Non-mammalian speciesshould also possess structurally or functionally related genes andproteins, e.g., birds or amphibians.

Solubility of a polypeptide or fragment depends upon the environment andthe polypeptide. Many parameters affect polypeptide solubility,including temperature, electrolyte environment, size and molecularcharacteristics of the polypeptide, and nature of the solvent.Typically, the temperature at which the polypeptide is used ranges fromabout 4° C. to about 65° C. Usually the temperature at use is greaterthan about 18° C. For diagnostic purposes, the temperature will usuallybe about room temperature or warmer, but less than the denaturationtemperature of components in the assay. For therapeutic purposes, thetemperature will usually be body temperature, typically about 37° C. forhumans and mice, though under certain situations the temperature may beraised or lowered in situ or in vitro.

The size and structure of the polypeptide should generally be in asubstantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents in a manner which approximates natural lipid bilayerinteractions. In certain contexts, e.g., Western blots, the protein willbe denatured, and/or attached to a solid substrate, e.g., in an affinitycolumn.

The solvent and electrolytes will usually be a biologically compatiblebuffer, of a type used for preservation of biological activities, andwill usually approximate a physiological aqueous solvent. Usually thesolvent will have a neutral pH, typically between about 5 and 10, andpreferably about 7.5. On some occasions, one or more detergents will beadded, typically a mild non-denaturing one, e.g., CHS (cholesterylhemisuccinate) or CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), or a low enough concentration as to avoid significantdisruption of structural or physiological properties of the protein.

A. Physical Variants

This invention also encompasses proteins or peptides having substantialamino acid sequence identity with the amino acid sequence of the 312C2.The variants include species, polymorphic, or allelic variants.

Amino acid sequence homology, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453;Sankoff, et al. (1983) Chapter One in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group, Madison, Wis. Sequence identity changes whenconsidering conservative substitutions as matches. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine, valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. Homologous amino acid sequencesare typically intended to include natural polymorphic or allelic andinterspecies variations in each respective protein sequence. Typicalhomologous proteins or peptides will have from 25-100% identity (if gapscan be introduced), to 50-100% identity (if conservative substitutionsare included) with the amino acid sequence of the 312C2. Identitymeasures will be at least about 35%, generally at least about 40%, oftenat least about 50%, typically at least about 60%, usually at least about70%, preferably at least about 80%, and more preferably at least about90%.

The isolated 312C2 DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, andinversions of nucleotide stretches. These modifications result in novelDNA sequences which encode these antigens, their derivatives, orproteins having similar physiological, immunogenic, antigenic, or otherfunctional activity. These modified sequences can be used to producemutant antigens or to enhance expression. Enhanced expression mayinvolve gene amplification, increased transcription, increasedtranslation, and other mechanisms. “Mutant 312C2” encompasses apolypeptide otherwise falling within the sequence identity definition ofthe 312C2 as set forth above, but having an amino acid sequence whichdiffers from that of 312C2 as normally found in nature, whether by wayof deletion, substitution, or insertion. This generally includesproteins having significant identity with a protein having sequence ofSEQ ID NO: 2, and as sharing various biological activities, e.g.,antigenic or immunogenic, with those sequences, and in preferredembodiments contain most of the full length disclosed sequences.Preferred variants will share a plurality of immune epitopes with therecited sequences, or equivalent proteins. Full length sequences willtypically be preferred, though truncated versions will also be useful,likewise, genes or proteins found from natural sources are typicallymost desired. Similar concepts apply to different 312C2 proteins,particularly those found in various warm blooded animals, e.g., mammalsand birds. These descriptions are generally meant to encompass all 312C2proteins, not limited to the particular mouse or human embodimentsspecifically discussed.

312C2 mutagenesis can also be conducted by making amino acid insertionsor deletions. Substitutions, deletions, insertions, or any combinationsmay be generated to arrive at a final construct. Insertions includeamino- or carboxy-terminal fusions. Random mutagenesis can be conductedat a target codon and the expressed mutants can then be screened for thedesired activity. Methods for making substitution mutations atpredetermined sites in DNA having a known sequence are well known in theart, e.g., by M13 primer mutagenesis or polymerase chain reaction (PCR)techniques. See, e.g., Sambrook, et al. (1989); Ausubel, et al. (1987and Supplements); and Kunkel, et al. (1987) Methods in Enzymol.154:367-382.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. A similar conceptapplies to heterologous nucleic acid sequences.

In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, target-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence, e.g., PCRtechniques.

B. Functional Variants

The blocking of physiological response to 312C2s may result from theinhibition of binding of the antigen to its binding partner, e.g.,another of itself, likely through competitive inhibition. Thus, in vitroassays of the present invention will often use isolated protein,membranes from cells expressing a membrane associated recombinant 312C2,soluble fragments comprising antigen binding segments of these proteins,or fragments attached to solid phase substrates. These assays will alsoallow for the diagnostic determination of the effects of either bindingsegment mutations and modifications, or antigen mutations andmodifications, e.g., 312C2 analogs. Structure-activity analysis will beperformed with mutational variants.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to antigen or bindingfragments compete with a test compound for binding to the protein, e.g.,of natural protein sequence.

“Derivatives” of 312C2 antigens include amino acid sequence mutants fromnaturally occurring forms, glycosylation variants, and covalent oraggregate conjugates with other chemical moieties. Covalent derivativescan be prepared by linkage of functionalities to groups which are foundin 312C2 amino acid side chains or at the N- or C-termini, e.g., bystandard means. See, e.g., Lundblad and Noyes (1988) Chemical Reagentsfor Protein Modification, vols. 1-2, CRC Press, Inc., Boca Raton, Fla.;Hugli (ed.) (1989) Techniques in Protein Chemistry, Academic Press, SanDiego, Calif.; and Wong (1991) Chemistry of Protein Conjugation andCross Linking, CRC Press, Boca Raton, Fla.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. See, e.g.,Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versionsof the peptides with the same primary amino acid sequence which haveother minor modifications, including phosphorylated amino acid residues,e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Fusion polypeptides between 312C2s and other homologous or heterologousproteins are also provided. Many cytokine receptors or other surfaceproteins are multimeric, e.g., homodimeric entities, and a repeatconstruct may have various advantages, including lessened susceptibilityto proteolytic cleavage. Typical examples are fusions of a reporterpolypeptide, e.g., luciferase, with a segment or domain of a protein,e.g., a receptor-binding segment, so that the presence or location ofthe fused ligand may be easily determined. See, e.g., Dull, et al., U.S.Pat. No. 4,859,609. Other gene fusion partners include bacterialβ-galactosidase, trpE, Protein A, β-lactamase, alpha amylase, alcoholdehydrogenase, yeast alpha mating factor, and detection or purificationtags such as a FLAG sequence of His6 sequence. See, e.g., Godowski, etal. (1988) Science 241:812-816.

Fusion peptides will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, e.g., inSambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.),vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al. (eds.)(1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.Techniques for synthesis of polypeptides are described, e.g., inMerrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986)Science 232:341-347; Atherton, et al. (1989) Solid Phase PeptideSynthesis: A Practical Approach, IRL Press, Oxford; and Grant (1992)Synthetic Peptides: A User's Guide, W.H. Freeman, NY.

This invention also contemplates the use of derivatives of 312C2s otherthan variations in amino acid sequence or glycosylation. Suchderivatives may involve covalent or aggregative association withchemical moieties. Covalent or aggregative derivatives will be useful asimmunogens, as reagents in immunoassays, or in purification methods suchas for affinity purification of binding partners, e.g., other antigens.A 312C2 can be immobilized by covalent bonding to a solid support suchas cyanogen bromide-activated SEPHAROSE, by methods which are well knownin the art, or adsorbed onto polyolefin surfaces, with or withoutglutaraldehyde cross-linking, for use in the assay or purification ofanti-312C2 antibodies or an alternative binding composition. Westernblot techniques are also common. The 312C2s can also be labeled with adetectable group, e.g., for use in diagnostic assays. Purification of312C2 may be effected by an immobilized antibody or complementarybinding partner.

A solubilized 312C2 or fragment of this invention can be used as animmunogen for the production of antisera or antibodies specific forbinding to the antigen or fragments thereof. Purified antigen can beused to screen or purify monoclonal antibodies or antigen-bindingfragments, encompassing antigen binding fragments of natural antibodies,e.g., Fab, Fab′, F(ab)₂, etc. Purified 312C2s can also be used as areagent to detect antibodies generated in response to the presence ofelevated levels of the antigen or cell fragments containing the antigen,both of which may be diagnostic of an abnormal or specific physiologicalor disease condition. This invention contemplates antibodies raisedagainst amino acid sequences encoded by nucleotide sequence shown in SEQID NO: 1, or fragments of proteins containing it. In particular, thisinvention contemplates antibodies having binding affinity to or beingraised against specific fragments which are predicted to lie outside ofthe lipid bilayer, both extracellular or intracellular.

The present invention contemplates the isolation of additional closelyrelated species variants. Southern and Northern blot analysis shouldestablish that similar genetic entities exist in other mammals. It islikely that 312C2s are widespread in species variants, e.g., rodents,lagomorphs, carnivores, artiodactyla, perissodactyla, and primates.

The invention also provides means to isolate a group of related antigensdisplaying both distinctness and similarities in structure, expression,and function. Elucidation of many of the physiological effects of themolecules will be greatly accelerated by the isolation andcharacterization of additional distinct species variants of them. Inparticular, the present invention provides useful probes for identifyingadditional homologous genetic entities in different species.

The isolated genes will allow transformation of cells lacking expressionof a corresponding 312C2, e.g., either species types or cells which lackcorresponding antigens and exhibit negative background activity. Thisshould allow analysis of the function of 312C2 in comparison tountransformed control cells.

Dissection of critical structural elements which effect the variousactivation or differentiation functions mediated through these antigensis possible using standard techniques of modern molecular biology,particularly in comparing members of the related class. See, e.g., thehomolog-scanning mutagenesis technique described in Cunningham, et al.(1989) Science 243:1339-1336; and approaches used in O'Dowd, et al.(1988) J. Biol. Chem. 263:15985-15992; and Lechleiter, et al. (1990)EMBO J. 9:4381-4390. Structure activity relationship can be analyzedusing variants.

Intracellular functions would probably involve segments of the antigenwhich are normally accessible to the cytosol. However, proteininternalization may occur under certain circumstances, and interactionbetween intracellular components and “extracellular” segments may occur.The specific segments of interaction of 312C2 with other intracellularcomponents may be identified by mutagenesis or direct biochemical means,e.g., cross-linking or affinity methods. Structural analysis bycrystallographic or other physical methods will also be applicable.Further investigation of the mechanism of signal transduction willinclude study of associated components which may be isolatable byaffinity methods or by genetic means, e.g., complementation analysis ofmutants.

Further study of the expression and control of 312C2 will be pursued.The controlling elements associated with the antigens should exhibitdifferential physiological, developmental, tissue specific, or otherexpression patterns. Upstream or downstream genetic regions, e.g.,control elements, are of interest. In particular, physiological ordevelopmental variants, e.g., multiple alternatively processed forms ofthe mouse antigen have been found. See, e.g., SEQ ID NO: 1. Thus,differential splicing of message may lead to an assortment of membranebound forms, soluble forms, and modified versions of antigen.

With human 312C2, 6 alternatively processed forms have been isolated.Clone A8, a truncated form of 312C2, is missing 7 amino acidsimmediately after the transmembrane domain. See SEQ ID NO: 6. Clone A5is identical to 312C2 for the first 105 amino acids. It is believed thatthe divergence may be due to an unspliced intron. See SEQ ID NO: 7.Clone G10 is identical to 312C2 for the first 202 amino acids, but thenvaries in the 11 amino acids after the transmembrane domain and is 76amino acids longer in the intracellular domain. The intracellular domainof G10, like that of 312C2, does not contain a death domain. See SEQ IDNO: 8.

Structural studies of the antigens will lead to design of new antigens,particularly analogs exhibiting agonist or antagonist properties on themolecule. This can be combined with previously described screeningmethods to isolate antigens exhibiting desired spectra of activities.

IV. Antibodies

Antibodies can be raised to various 312C2s, including species,polymorphic, or allelic variants, and fragments thereof, both in theirnaturally occurring forms and in their recombinant forms. Additionally,antibodies can be raised to 312C2s in either their active forms or intheir inactive forms, including native or denatured versions.Anti-idiotypic antibodies are also contemplated.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the antigens can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective 312C2s, or screened for agonistic orantagonistic activity, e.g., mediated through the antigen or its bindingpartner. Antibodies may be agonistic or antagonistic, e.g., bysterically blocking ligand binding. These monoclonal antibodies willusually bind with at least a K_(D) of about 1 mM, more usually at leastabout 300 μM, typically at least about 100 μM, more typically at leastabout 30 μM, preferably at least about 10 μM, and more preferably atleast about 3 μM or better.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for ability to bind to the antigens without inhibiting bindingby a partner. As neutralizing antibodies, they can be useful incompetitive binding assays. They will also be useful in detecting orquantifying 312C2 protein or its binding partners. See, e.g., Chan (ed.)(1987) Immunology: A Practical Guide, Academic Press, Orlando, Fla.;Price and Newman (eds.) (1991) Principles and Practice of Immunoassay,Stockton Press, N.Y.; and Ngo (ed.) (1988) Nonisotopic Immunoassay,Plenum Press, N.Y. Cross absorptions or other tests will identifyantibodies which exhibit various spectra of specificities, e.g., uniqueor shared species specificities.

Further, the antibodies, including antigen binding fragments, of thisinvention can be potent antagonists that bind to the antigen and inhibitfunctional binding or inhibit the ability of a binding partner to elicita biological response. They also can be useful as non-neutralizingantibodies and can be coupled to toxins or radionuclides so that whenthe antibody binds to antigen, a cell expressing it, e.g., on itssurface, is killed. Further, these antibodies can be conjugated to drugsor other therapeutic agents, either directly or indirectly by means of alinker, and may effect drug targeting.

Antigen fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. An antigen and its fragments may be fused or covalentlylinked to a variety of immunogens, such as keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, etc. See Microbiology, HoeberMedical Division, Harper and Row, 1969; Landsteiner (1962) Specificityof Serological Reactions, Dover Publications, New York; Williams, et al.(1967) Methods in Immunology and Immunochemistry, vol. 1, AcademicPress, New York; and Harlow and Lane (1988) Antibodies: A LaboratoryManual, CSH Press, NY, for descriptions of methods of preparingpolyclonal antisera.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual,CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.), Academic Press, New York; and particularly in Kohler andMilstein (1975) in Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567; Moore, et al.,U.S. Pat. No. 4,642,334; and Queen, et al. (1989) Proc. Nat'l Acad. Sci.USA 86:10029-10033.

The antibodies of this invention can also be used for affinitychromatography in isolating the protein. Columns can be prepared wherethe antibodies are linked to a solid support. See, e.g., Wilchek et al.(1984) Meth. Enzymol. 104:3-55.

Antibodies raised against each 312C2 will also be useful to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

V. Nucleic Acids

The described peptide sequences and the related reagents are useful indetecting, isolating, or identifying a DNA clone encoding 312C2, e.g.,from a natural source. Typically, it will be useful in isolating a genefrom mammal, and similar procedures will be applied to isolate genesfrom other species, e.g., warm blooded animals, such as birds andmammals. Cross hybridization will allow isolation of 312C2 from otherspecies. A number of different approaches should be available tosuccessfully isolate a suitable nucleic acid clone.

The purified protein or defined peptides are useful for generatingantibodies by standard methods, as described above. Synthetic peptidesor purified protein can be presented to an immune system to generatemonoclonal or polyclonal antibodies. See, e.g., Coligan (1991) CurrentProtocols in Immunology Wiley/Greene; and Harlow and Lane (1989)Antibodies: A Laboratory Manual, Cold Spring Harbor Press.Alternatively, the 312C2 can be used as a specific binding reagent, andadvantage can be taken of its specificity of binding, much like anantibody would be used.

For example, the specific binding composition could be used forscreening of an expression library made from a cell line which expressesa 312C2. The screening can be standard staining of surface expressedantigen, or by panning. Screening of intracellular expression can alsobe performed by various staining or immunofluorescence procedures. Thebinding compositions could be used to affinity purify or sort out cellsexpressing the protein.

The peptide segments can also be used to predict appropriateoligonucleotides to screen a library. The genetic code can be used toselect appropriate oligonucleotides useful as probes for screening. See,e.g., SEQ ID NO: 1 or 3. In combination with polymerase chain reaction(PCR) techniques, synthetic oligonucleotides will be useful in selectingcorrect clones from a library. Complementary sequences will also be usedas probes, primers, or antisense strands. Based upon identification ofthe likely extracellular domain, various fragments should beparticularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides.

This invention contemplates use of isolated DNA or fragments to encode abiologically active corresponding 312C2 polypeptides. See SEQ ID NO: 5.In addition, this invention covers isolated or recombinant DNA whichencodes a biologically active, e.g., antigenic, protein or polypeptidewhich is capable of hybridizing under appropriate conditions with theDNA sequences described herein. Said biologically active protein orpolypeptide can be an intact antigen, or fragment, and have an aminoacid sequence disclosed in, e.g., SEQ ID NO: 2 or 4. Further, thisinvention covers the use of isolated or recombinant DNA, or fragmentsthereof, which encode proteins which are homologous to a 312C2 or whichwas isolated using cDNA encoding a 312C2 as a probe. The isolated DNAcan have the respective regulatory sequences in the 5′ and 3′ flanks,e.g., promoters, enhancers, poly-A addition signals, and others.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially separated from other componentswhich naturally accompany a native sequence, e.g., ribosomes,polymerases, and/or flanking genomic sequences from the originatingspecies. The term embraces a nucleic acid sequence which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.Generally, the nucleic acid will be in a vector or fragment less thanabout 50 kb, usually less than about 30 kb, typically less than about 10kb, and preferably less than about 6 kb.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain minor heterogeneity.This heterogeneity is typically found at the polymer ends or portionsnot critical to a desired biological function or activity.

A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, e.g., products made by transforming cells with anyunnaturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site.

Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a single genetic entity comprising adesired combination of functions not found in the commonly availablenatural forms. Restriction enzyme recognition sites are often the targetof such artificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design. Asimilar concept is intended for a recombinant, e.g., fusion,polypeptide. Specifically included are synthetic nucleic acids which, bygenetic code redundancy, encode polypeptides similar to fragments ofthese antigens, and fusions of sequences from various different speciesvariants. Alternatively, a heterologous promoter may be insertedupstream from a natural gene.

A significant “fragment” in a nucleic acid context is a contiguoussegment of at least about 17 nucleotides, generally at least about 22nucleotides, ordinarily at least about 29 nucleotides, more often atleast about 35 nucleotides, typically at least about 41 nucleotides,usually at least about 47 nucleotides, preferably at least about 55nucleotides, and in particularly preferred embodiments will be at leastabout 60 or more nucleotides.

A DNA which codes for a 312C2 protein will be particularly useful toidentify genes, mRNA, and cDNA species which code for related orhomologous proteins, as well as DNAs which code for homologs proteinsfrom different species. There are likely homologues in other species,including primates, rodents, and birds. Various 312C2 proteins should behomologous and are encompassed herein. However, even proteins that havea more distant evolutionary relationship to the antigen can readily beisolated under appropriate conditions using these sequences if they aresufficiently homologous. Primate 312C2 proteins are of particularinterest.

Recombinant clones derived from the genomic sequences, e.g., containingintrons, will be useful for transgenic studies, including, e.g.,transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow(1992) “Transgenic Animals” in Roitt (ed.) Encyclopedia of Immunology,Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science256:1392-1394; Kuhn, et al. (1991) Science 254:707-710; Capecchi (1989)Science 244:1288; Robertson (1987)(ed.) Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, IRL Press, Oxford; and Rosenberg(1992) J. Clinical Oncology 10:180-199.

Substantial homology in the nucleic acid sequence comparison contextmeans either that the segments, or their complementary strands, whencompared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 50% of thenucleotides, generally at least about 58%, ordinarily at least about65%, often at least about 71%, typically at least about 77%, usually atleast about 85%, preferably at least about 95 to 98% or more, and inparticular embodiments, as high as about 99% or more of the nucleotides.Alternatively, substantial homology exists when the segments willhybridize under selective hybridization conditions, to a strand, or itscomplement, typically using a sequence of 312C2, e.g., in SEQ ID NO: 1or 3. Typically, selective hybridization will occur when there is atleast about 55% homology over a stretch of at least about 30nucleotides, preferably at least about 75% over a stretch of about 25nucleotides, and most preferably at least about 90% over about 20nucleotides. See, Kanehisa (1984) Nuc. Acids Res. 12:203-213. The lengthof homology comparison, as described, may be over longer stretches, andin certain embodiments will be over a stretch of at least about 17nucleotides, usually at least about 28 nucleotides, typically at leastabout 40 nucleotides, and preferably at least about 75 to 100 or morenucleotides.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters, typically those controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., usually in excess ofabout 37° C., typically in excess of about 55° C., preferably in excessof about 70° C. Stringent salt conditions will ordinarily be less thanabout 1000 mM, usually less than about 400 mM, typically less than about250 mM, preferably less than about 150 mM. However, the combination ofparameters is much more important than the measure of any singleparameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.31:349-370.

312C2 from other mammalian species can be cloned and isolated bycross-species hybridization of closely related species. Homology may berelatively low between distantly related species, and thus hybridizationof relatively closely related species is advisable. Alternatively,preparation of an antibody preparation which exhibits less speciesspecificity may be useful in expression cloning approaches.

VI. Making 312C2; Mimetics

DNA which encodes the 312C2 or fragments thereof can be obtained bychemical synthesis, screening cDNA libraries, or screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.See, e.g., Okayama and Berg (1982) Mol. Cell. Biol. 2:161-170; Gublerand Hoffman (1983) Gene 25:263-269; and Glover (ed.) (1984) DNA Cloning:A Practical Approach, IRL Press, Oxford. Alternatively, the sequencesprovided herein provide useful PCR primers or allow synthetic or otherpreparation of suitable genes encoding a 312C2; including, naturallyoccurring embodiments.

This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length 312C2 or fragments which can in turn, e.g.,be used to generate polyclonal or monoclonal antibodies; for bindingstudies; for construction and expression of modified molecules; and forstructure/function studies.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. See, e.g.,Pouwels, et al. (1985 and Supplements) Cloning Vectors: A LaboratoryManual, Elsevier, N.Y.; and Rodriguez, et al. (1988)(eds.) Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Buttersworth,Boston, Mass.

For purposes of this invention, DNA sequences are operably linked whenthey are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression. See e.g., Rodriguez, et al., Chapter 10, pp. 205-236; Balbasand Bolivar (1990) Methods in Enzymology 185:14-37; and Ausubel, et al.(1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.

Representative examples of suitable expression vectors include pCDNA1;pCD, see Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142; pMC1neoPoly-A, see Thomas, et al. (1987) Cell 51:503-512; and a baculovirusvector such as pAC 373 or pAC 610. See, e.g., Miller (1988) Ann. Rev.Microbiol. 42:177-199.

It will often be desired to express a 312C2 polypeptide in a systemwhich provides a specific or defined glycosylation pattern. See, e.g.,Luckow and Summers (1988) Bio/Technology 6:47-55; and Kaufman (1990)Meth. Enzymol. 185:487-511.

The 312C2, or a fragment thereof, may be engineered to be phosphatidylinositol (PI) linked to a cell membrane, but can be removed frommembranes by treatment with a phosphatidyl inositol cleaving enzyme,e.g., phosphatidyl inositol phospholipase-C. This releases the antigenin a biologically active form, and allows purification by standardprocedures of protein chemistry. See, e.g., Low (1989) Biochim. Biophys.Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner,et al. (1991) J. Cell Biol. 114:1275-1283.

Now that the 312C2 has been characterized, fragments or derivativesthereof can be prepared by conventional processes for synthesizingpeptides. These include processes such as are described in Stewart andYoung (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co.,Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice of PeptideSynthesis, Springer-Verlag, New York; Bodanszky (1984) The Principles ofPeptide Synthesis, Springer-Verlag, New York; and Villafranca (ed.)(1991) Techniques in Protein Chemistry II, Academic Press, San Diego,Ca.

VII. Uses

The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in thegeneral description for T cell mediated conditions, or below in thedescription of kits for diagnosis. The antigen is useful as a marker,e.g., to identify T or NK cell subsets, or as a positive selectionmarker to fractionate immune subsets.

This invention also provides reagents with significant therapeuticvalue. The 312C2 (naturally occurring or recombinant), fragmentsthereof, and antibodies thereto, along with compounds identified ashaving binding affinity to 312C2, should be useful in the treatment ofconditions associated with abnormal physiology or development, includingabnormal proliferation, e.g., cancerous conditions, or degenerativeconditions. In particular, modulation of development of lymphoid cellswill be achieved by appropriate therapeutic treatment using thecompositions provided herein. For example, a disease or disorderassociated with abnormal expression or abnormal signaling by a 312C2should be a likely target for an agonist or antagonist of the antigen.The antigen plays a role in regulation or development of hematopoieticcells, e.g., lymphoid cells, which affect immunological responses, e.g.,autoimmune disorders.

In particular, the antigen will likely provide a costimulatory signal toT cell activation. Thus, the 312C2 will likely mediate T cellinteractions with other cell types. These interactions lead, inparticular contexts, to cell proliferation, enhanced cytokine synthesisby the cells, and consequential amplification of T cell proliferation.

Moreover, the 312C2 or antagonists could redirect T cell responses,e.g., towards a Th0/Th1 pathway, or towards a Th2 type response. Amongthese agonists should be various antibodies which recognize theappropriate epitopes, e.g., which mimic binding of 312C2 to its ligand.Alternatively, antibody antagonists may bind to epitopes whichsterically can block partner binding.

Conversely, antagonists of 312C2, such as the naturally occurringsecreted form of 312C2 or blocking antibodies, may provide a selectiveand powerful way to block immune responses in abnormal situations, e.g.,autoimmune disorders, including rheumatoid arthritis, systemic lupuserythrematosis (SLE), Hashimoto's autoimmune thyroiditis, as well asacute and chronic inflammatory responses in which T cell activation,expansion, and/or immunological T cell memory play an important role.See also Samter, et al. (eds.) Immunological Diseases vols. 1 and 2,Little, Brown and Co. Suppression of T cell activation, expansion,and/or cytokine release by the naturally occurring secreted form of312C2, which can be produced in large quantities by recombinant methods,or by blocking antibodies, should be effective in many disorders inwhich abnormal or undesired T cell responses are of importance, e.g., ina transplantation rejection situation.

In addition, certain combination compositions with other modulators of Tcell signaling would be useful. Such other signaling molecules includeTcR reagents, CD40, CD40L, CTLA-8, CD28, SLAM, FAS, and their respectiveantagonists.

Various abnormal conditions are known in each of the cell types shown topossess 312C2 mRNA by Northern blot analysis. See Berkow (ed.) The MerckManual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; Thorn, etal. Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y.; andWeatherall, et al. (eds.) Oxford Textbook of Medicine, Oxford UniversityPress, Oxford. Many other medical conditions and diseases involve Tcells or are T cell mediated, and many of these will be responsive totreatment by an agonist or antagonist provided herein. See, e.g., Stitesand Terr (eds; 1991) Basic and Clinical Immunology Appleton and Lange,Norwalk, Conn.; and Samter, et al. (eds) Immunological Diseases Little,Brown and Co. These problems should be susceptible to prevention ortreatment using compositions provided herein.

312C2 antibodies can be purified and then administered to a patient,veterinary or human. These reagents can be combined for therapeutic usewith additional active or inert ingredients, e.g., in conventionalpharmaceutically acceptable carriers or diluents, e.g., immunogenicadjuvants, along with physiologically innocuous stabilizers, excipients,buffers, or preservatives. These combinations can be sterile filteredand placed into dosage forms as by lyophilization in dosage vials orstorage in stabilized aqueous preparations. This invention alsocontemplates use of antibodies or binding fragments thereof, includingforms which are not complement binding. Sterile compositions of nucleicacids and proteins are also contemplated.

Drug screening using 312C2 or fragments thereof can be performed toidentify compounds having binding affinity to or other relevantbiological effects on 312C2 functions, including isolation of associatedcomponents. Subsequent biological assays can then be utilized todetermine if the compound has intrinsic stimulating activity and istherefore a blocker or antagonist in that it blocks the activity of theantigen. Likewise, a compound having intrinsic stimulating activity canactivate the signal pathway and is thus an agonist in that it simulatesthe activity of 312C2. This invention further contemplates thetherapeutic use of blocking antibodies to 312C2 as antagonists and ofstimulatory antibodies, e.g., A12, as agonists. This approach should beparticularly useful with other 312C2 species variants.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17thed. (1990), Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 pM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous or long term administration. See, e.g., Langer (1990) Science249:1527-1533.

312C2, fragments thereof, and antibodies to it or its fragments,antagonists, and agonists, may be administered directly to the host tobe treated or, depending on the size of the compounds, it may bedesirable to conjugate them to carrier proteins such as ovalbumin orserum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, topical, or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. See, e.g., Gilman, et al.(eds.) (1990) Goodman and Gilman's: The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, etal. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications,Dekker, New York; Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Tablets, Dekker, New York; and Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York. Thetherapy of this invention may be combined with or used in associationwith other agents, e.g., other modulators of T cell activation, e.g.,CD40, CD40 ligand, CD28, CTLA-4, B7, B70, SLAM, T cell receptorsignaling entities, or their respective antagonists.

Both the naturally occurring and the recombinant form of the 312C2s ofthis invention are particularly useful in kits and assay methods whichare capable of screening compounds for binding activity to the proteins.Several methods of automating assays have been developed in recent yearsso as to permit screening of tens of thousands of compounds in a shortperiod. See, e.g., Fodor, et al. (1991) Science 251:767-773, whichdescribes means for testing of binding affinity by a plurality ofdefined polymers synthesized on a solid substrate. The development ofsuitable assays can be greatly facilitated by the availability of largeamounts of purified, soluble 312C2 as provided by this invention.

Other methods can be used to determine the critical residues in the312C2-312C2 ligand interactions. Mutational analysis can be performed,e.g., see Somoza, et al. (1993) J. Exptl. Med. 178:549-558, to determinespecific residues critical in the interaction and/or signaling. Bothextracellular domains, involved in the homophilic interaction, orintracellular domain, which provides interactions important inintracellular signaling.

For example, antagonists can normally be found once the antigen has beenstructurally defined, e.g., by tertiary structure data. Testing ofpotential interacting analogs is now possible upon the development ofhighly automated assay methods using a purified 312C2. In particular,new agonists and antagonists will be discovered by using screeningtechniques described herein. Of particular importance are compoundsfound to have a combined binding affinity for a spectrum of 312C2molecules, e.g., compounds which can serve as antagonists for speciesvariants of 312C2.

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing a 312C2. Cells may be isolated which express a 312C2 inisolation from other molecules. Such cells, either in viable or fixedform, can be used for standard binding partner binding assays. See also,Parce, et al. (1989) Science 246:243-247; and Owicki, et al. (1990)Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto a 312C2 and is described in detail in Geysen, European PatentApplication 84/03564, published on Sep. 13, 1984. First, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, e.g., plastic pins or some other appropriate surface, seeFodor, et al. (1991). Then all the pins are reacted with solubilized,unpurified or solubilized, purified 312C2, and washed. The next stepinvolves detecting bound 312C2.

Rational drug design may also be based upon structural studies of themolecular shapes of the 312C2 and other effectors or analogs. Effectorsmay be other proteins which mediate other functions in response tobinding, or other proteins which normally interact with 312C2. One meansfor determining which sites interact with specific other proteins is aphysical structure determination, e.g., x-ray crystallography or 2dimensional NMR techniques. These will provide guidance as to whichamino acid residues form molecular contact regions. For a detaileddescription of protein structural determination, see, e.g., Blundell andJohnson (1976) Protein Crystallography, Academic Press, New York.Structure from related TcR family genes will also provide furtherinsight.

VIII. Kits

This invention also contemplates use of 312C2 proteins, fragmentsthereof, peptides, and their fusion products in a variety of diagnostickits and methods for detecting the presence of another 312C2 or bindingpartner. Typically the kit will have a compartment containing either adefined 312C2 peptide or gene segment or a reagent which recognizes oneor the other, e.g., 312C2 fragments or antibodies.

A kit for determining the binding affinity of a test compound to a 312C2would typically comprise a test compound; a labeled compound, forexample a binding partner or antibody having known binding affinity for312C2; a source of 312C2 (naturally occurring or recombinant); and ameans for separating bound from free labeled compound, such as a solidphase for immobilizing the molecule. Once compounds are screened, thosehaving suitable binding affinity to the antigen can be evaluated insuitable biological assays, as are well known in the art, to determinewhether they act as agonists or antagonists to the signaling pathway.The availability of recombinant 312C2 polypeptides also provide welldefined standards for calibrating such assays. Histological analysis isalso possible.

A preferred kit for determining the concentration of, e.g., a 312C2 in asample would typically comprise a labeled compound, e.g., bindingpartner or antibody, having known binding affinity for the antigen, asource of antigen (naturally occurring or recombinant) and a means forseparating the bound from free labeled compound, e.g., a solid phase forimmobilizing the 312C2. Compartments containing reagents, andinstructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for the 312C2or fragments are useful in diagnostic applications to detect thepresence of elevated levels of 312C2 and/or its fragments. Suchdiagnostic assays can employ lysates, live cells, fixed cells,immunofluorescence, cell cultures, body fluids, and further can involvethe detection of antigens related to the antigen in serum, or the like.Diagnostic assays may be homogeneous (without a separation step betweenfree reagent and antigen-binding partner complex) or heterogeneous (witha separation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like.See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525; Harlow andLane (1980) Antibodies: A Laboratory Manual, CSH Press, NY; and Coligan,et al. (eds.) (1993) Current Protocols in Immunology, Greene and Wiley,NY.

Anti-idiotypic antibodies may have similar use to diagnose presence ofantibodies against a 312C2, as such may be diagnostic of variousabnormal states. For example, overproduction of 312C2 may result inproduction of various immunological reactions which may be diagnostic ofabnormal physiological states, particularly in proliferative cellconditions such as cancer or abnormal activation or differentiation.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or binding partner, or labeled312C2 is provided. This is usually in conjunction with other additives,such as buffers, stabilizers, materials necessary for signal productionsuch as substrates for enzymes, and the like. Preferably, the kit willalso contain instructions for proper use and disposal of the contentsafter use. Typically the kit has compartments for each useful reagent.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium providingappropriate concentrations of reagents for performing the assay. Kitsmay be for solution determination, or histology in tissue samples.

Many of the aforementioned constituents of the drug screening and thediagnostic assays may be used without modification or may be modified ina variety of ways. For example, labeling may be achieved by covalentlyor non-covalently joining a moiety which directly or indirectly providesa detectable signal. In any of these assays, the binding partner, testcompound, 312C2, or antibodies thereto can be labeled either directly orindirectly. Possibilities for direct labeling include label groups:radiolabels such as ¹²⁵I, enzymes (U.S. Pat. No. 3,645,090) such asperoxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat.No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups.

There are also numerous methods of separating the bound from the free312C2, or alternatively the bound from the free test compound. The 312C2can be immobilized on various matrixes followed by washing. Suitablematrixes include plastic such as an ELISA plate, filters, and beads.See, e.g., Coligan, et al. (eds.) (1993) Current Protocols inImmunology, Vol. 1, Chapter 2, Greene and Wiley, NY. Other suitableseparation techniques include, without limitation, the fluoresceinantibody magnetizable particle method described in Rattle, et al. (1984)Clin. Chem. 30:1457-1461, and the double antibody magnetic particleseparation as described in U.S. Pat. No. 4,659,678.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence of a312C2. These sequences can be used as probes for detecting levels of the312C2 message in samples from patients suspected of having an abnormalcondition, e.g., cancer or developmental problem. Since the antigen is amarker for activation, it may be useful to determine the numbers ofactivated T cells to determine, e.g., when additional suppression may becalled for. The preparation of both RNA and DNA nucleotide sequences,the labeling of the sequences, and the preferred size of the sequenceshas received ample description and discussion in the literature. See,e.g., Langer-Safer, et al. (1982) Proc. Nat'l. Acad. Sci. 79:4381-4385;Caskey (1987) Science 236:962-967; and Wilchek et al. (1988) Anal.Biochem. 171:1-32. Histological analysis may also be performed.

Diagnostic kits which also test for the qualitative or quantitativepresence of other markers are also contemplated. Diagnosis or prognosismay depend on the combination of multiple indications used as markers.Thus, kits may test for combinations of markers. See, e.g., Viallet, etal. (1989) Progress in Growth Factor Res. 1:89-97. Other kits may beused to evaluate T cell subsets, e.g., analysis or isolation, usingconservative or destructive means.

IX. Methods for Isolating 312C2 Specific Binding Partners

The 312C2 protein should interact with a ligand based, e.g., upon itssimilarity in structure and function to other cell surface antigensexhibiting similar structure and cell type specificity of expression.Methods to isolate a ligand are made available by the ability to makepurified 312C2 for screening programs. Soluble or other constructs usingthe 312C2 sequences provided herein will allow for screening orisolation of 312C2 specific ligands. Many methods exist for expressioncloning, panning, affinity isolation, cross-linking, genetic selection,or other means to identify a receptor ligand.

A variety of different assays for detecting compounds capable of bindingto 312C2 are used in the present invention. For instance, the binding ofa test compound to 312C2 or a peptide fragment thereof can be measureddirectly, in the presence or absence of 312C2 polypeptide. This lattertype of assay is called a direct binding assay. In addition, compoundswhich inhibit the binding of 312C2 to specific, preferably monoclonal,antibodies can be identified in competitive binding assays. Both directbinding assays and competitive binding assays can be used in a varietyof different formats, similar to the formats used in immunoassays andreceptor binding assays. For a description of different formats forbinding assays, including competitive binding assays and direct bindingassays, see Basic and Clinical Immunology 7th Edition (D. Stites and A.Terr ed.) 1991; Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, BocaRaton, Fla. (1980); and “Practice and Theory of Enzyme Immunoassays,” P.Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology,Elsevier Science Publishers B.V. Amsterdam (1985), each of which isincorporated herein by reference.

In competitive binding assays, for example, the sample compound cancompete with a labeled analyte for specific binding sites on a bindingagent bound to a solid surface. In this type of format, the labeledanalyte can be labeled 312C2 and the binding agent can be an antibodybound to a solid phase. Alternatively, the labeled analyte can belabeled antibody and the binding agent can be a solid phase wild type312C2 or a fragment thereof. The concentration of labeled analyte boundto the capture agent is inversely proportional to the ability of a testcompound to compete in the binding assay. The amount of inhibition oflabeled analyte by the test compound depends on the binding assayconditions and on the concentrations of binding agent, labeled analyte,and test compound that are used. Under specified assay conditions, acompound is said to be capable of inhibiting the binding of 312C2 to aspecific antibody in a competitive binding assay, if the amount ofbinding of the labeled analyte to the binding agent is decreased by 50%or preferably 90% or more. When a direct binding assay format is used, atest compound is said to bind an 312C2 when the signal measured is twicethe background level or higher.

In a competitive binding assay, the sample compound competes withlabeled protein for binding to a specific binding agent. As describedabove, the binding agent may be bound to a solid surface to effectseparation of bound labeled protein from the unbound labeled protein.Alternately, the competitive binding assay may be conducted in liquidphase, and any of a variety of techniques known in the art may be usedto separate the bound labeled protein from the unbound labeled protein.Following separation, the amount of bound labeled protein is determined.The amount of protein present in the sample is inversely proportional tothe amount of labeled protein binding.

Alternatively, a homogeneous binding assay may be performed in which aseparation step is not needed. In these type of binding assays, thelabel on the protein is altered by the binding of the protein to itsspecific binding agent. This alteration in the labeled protein resultsin a decrease or increase in the signal emitted by label, so thatmeasurement of the label at the end of the binding assay allows fordetection or quantitation of the protein.

The binding assay formats described herein employ labeled assaycomponents. The label can be in a variety of forms. The label may becoupled directly or indirectly to the desired component of the assayaccording to methods well known in the art. A wide variety of labels maybe used. The component may be labeled by any one of several methods.Traditionally, a radioactive label incorporating ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P is used. Non-radioactive labels include ligands which bind tolabeled antibodies, fluorophores, chemiluminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabeled ligand. The choice of label depends on sensitivity required,ease of conjugation with the compound, stability requirements, andavailable instrumentation. For a review of various labeling or signalproducing systems which may be used, see U.S. Pat. No. 4,391,904, whichis incorporated herein by reference.

Alternatively, an expression library can be screened for specificbinding to 312C2, e.g., by cell sorting, or other screening to detectsubpopulations which express such a binding component. See, e.g., Ho, etal. (1993) Proc. Nat'l Acad. Sci. USA 90:11267-11271. Alternatively, apanning method may be used. See, e.g., Seed and Aruffo (1987) Proc.Nat'l Acad. Sci. USA 84:3365-3369. A two-hybrid selection system mayalso be applied making appropriate constructs with the available 312C2sequences. See, e.g., Fields and Song (1989) Nature 340:245-246.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the invention tospecific embodiments.

EXAMPLES General Methods

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed.), vols. 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene and Wiley, New York; Innis, etal. (eds.)(1990) PCR Protocols: A Guide to Methods and Applications,Academic Press, N.Y. Methods for protein purification include suchmethods as ammonium sulfate precipitation, column chromatography,electrophoresis, centrifugation, crystallization, and others. See, e.g.,Ausubel, et al. (1987 and periodic supplements); Deutscher (1990) “Guideto Protein Purification” in Methods in Enzymology vol. 182, and othervolumes in this series; and manufacturer's literature on use of proteinpurification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad,Richmond, Calif. Combination with recombinant techniques allow fusion toappropriate segments, e.g., to a FLAG sequence or an equivalent whichcan be fused via a protease-removable sequence. See, e.g., Hochuli(1989) Chemische Industrie 12:69-70; Hochuli (1990) “Purification ofRecombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.)Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;and Crowe, et al. (1992) QIAexpress: The High Level Expression & ProteinPurification System QIAGEN, Inc., Chatsworth, Calif. Cell culturetechniques are described in Doyle, et al. (eds.) (1994) Cell and TissueCulture: Laboratory Procedures, John Wiley and Sons, NY.

Standard immunological techniques are described, e.g., in Hertzenberg,et al. (eds. 1996) Weir's Handbook of Experimental Immunology vols 1-4,Blackwell Science; Coligan (1991) Current Protocols in ImmunologyWiley/Greene, NY; and Methods in Enzymology volumes. 70, 73, 74, 84, 92,93, 108, 116, 121, 132, 150, 162, and 163.

Assays for vascular biological activities are well known in the art.They will cover angiogenic and angiostatic activities in tumor, or othertissues, e.g., arterial smooth muscle proliferation (see, e.g., Koyoma,et al. (1996) Cell 87:1069-1078), monocyte adhesion to vascularepithelium (see McEvoy, et al. (1997) J. Exp. Med. 185:2069-2077), etc.See also Ross (1993) Nature 362:801-809; Rekhter and Gordon (1995) Am.J. Pathol. 147:668-677; Thyberg, et al. (1990) Athersclerosis10:966-990; and Gumbiner (1996) Cell 84:345-357.

Assays for neural cell biological activities are described, e.g., inWouterlood (ed. 1995) Neuroscience Protocols modules 10, Elsevier;Methods in Neurosciences Academic Press; and Neuromethods Humana Press,Totowa, N.J. Methodology of developmental systems is described, e.g., inMeisami (ed.) Handbook of Human Growth and Developmental Biology CRCPress; and Chrispeels (ed.) Molecular Techniques and Approaches inDevelopmental Biology Interscience.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y. Fluorescent labelingof appropriate reagents was performed by standard methods.

Example 1 Cloning of Mouse 312C2 Antibodies and Flow-Cytometric Sorting

αβTcR+CD4−CD8− (DN) Thymocytes were Sorted Using CD4/CD8a-PE andTcRαβ-FITC mAbs (PharMingen, San Diego, Calif.). See Zlotnik, et al.(1992) J. Immunol. 4:1211-1215. The sorted cells (approximately 5×10⁵)were stimulated on solid-phase anti-CD3 for 24 h and were then expandedand cultured in IL-2 (500 U/ml) and IL-7 (100 U/ml) for one week (toapproximately 1×10⁸ cells). Cells were either harvested after one weekin culture or stimulated again for 6 h on anti-CD3 and then harvested.See Kelner, et al. (1994) Science 266:1395-1399.

Construction of Directional cDNA Libraries

Poly (A)+ RNA from anti-CD3 stimulated αβDN thymocytes or unstimulatedabDN thymocytes was used to synthesize first strand cDNA by usingNotI/Oligo-dT primer (Gibco-BRL, Gaithersburg, Md.). Double-strandedcDNA was synthesized, ligated with BstXI adaptors, digested with NotI,size fractionated for >0.5 kilobase pairs (kb) and ligated into theNotI/BstXI sites of pJFE-14, a derivative of the pCDSRα vector. SeeTakebe, et al. Mol. Cell Biol. 8:466-472. Electro-competent E. coliDH10α cells (Gibco-BRL) were used for transformation. Total number ofindependent clones of the cDNA libraries were 1.2×10⁶ for stimulatedαβDN and 8×10⁵ for unstimulated αβDN thymocytes, respectively.

Library Subtraction

The PCR-based subtraction system developed by Wang and Brown (1991)Proc. Natl. Acad. Sci. USA 88:11505-11509, was modified to apply toplasmid cDNA libraries. A cDNA library specific for activated αβDNthymocytes was generated using 100 μg of the unstimulated αβDN cDNAlibrary DNA digested with XbaI, NotI, and ScaI as driver DNA and 5 μg ofthe stimulated αβDN cDNA library DNA as tracer DNA. Followingrestriction digestion, the driver DNA was treated with DNA polymeraseKlenow fragment to fill-in the restriction sites. After ethanolprecipitation, the DNA was dissolved in 100 μl of water, heat-denaturedand mixed with 100 μl (100 μg) of Photoprobe biotin (VectorLaboratories, Burlingame, Calif.). The driver DNA was then irradiatedwith a 270-W sunlamp on ice for 20 min. 50 μl more Photoprobe biotin wasadded and the biotinylation reaction was repeated. After butanolextraction, the photobiotinylated DNA (driver-U) wasethanol-precipitated and dissolved in 30 μl of 10 mM Tris-HCl and 1 mMEDTA, pH 8 (TE). As tracer DNA, 5 μg of stimulated αβDN cDNA wasdigested with XbaI and NotI; ethanol precipitated; and dissolved in 4 μlof TE (tracer-S). Tracer-S was mixed with 15 μl of driver-U, 1 μl (10μg) of E. coli tRNA (Sigma, St. Louis, Mo.), and 20 μl of 2×hybridization buffer (1.5 M NaCl, 10 mM EDTA, 50 mM HEPES, pH 7.5, 0.2%SDS), overlaid with mineral oil, and heat-denatured. The sample tube wasimmediately transferred into a 68° C. water bath and incubated for 20 h.The reaction mixture was then subjected to streptavidin treatmentfollowed by phenol/chloroform extraction. Subtracted DNA wasprecipitated, dissolved in 12 μl of TE, mixed with 8 μl of driver-U and20 μl of 2× hybridization buffer, and then incubated at 68° C. for 2 h.After streptavidin treatment, the remaining DNA was ligated with 250 ngof a purified XbaI/NotI fragment of pJFE-14 and then transformed intoelectro-competent E. coli cells to generate the activation specific αβDNsubtracted library (S1). 100 independent clones were randomly picked andscreened by hybridization using a cocktail of known cytokine cDNAs.Plasmid DNA's were prepared from clones that did not hybridize to thecytokine probes. These clones were grouped by insert size and furthercharacterized by DNA sequencing. Clones corresponding to the 312C2 wereisolated.

Example 2 Cellular Expression of Mouse 312C2

A probe specific for cDNA encoding mouse 312C2 was used to determinetissue distribution of the antigen. All probes were labeled by randompriming.

The results showed that 312C2 was expressed most abundantly in T cells,in particular, certain subsets of activated T cells. Thymus, spleen, andlymph node appeared to have more expression than other tissues.Expression levels are: thymus +; Th1 subset ++++; Th2 subset ++++,NK1.1+ T cells ++; αβ T cells ++; pro T cells +; CD4+ cells ++; CD8+cells ++; and activated spleen cells +. A message was also detected incertain pro-, pre-, and mature B cell lines. The signal in the followingcell types suggested that expression is very low to virtually absent inlung, heart, kidney, macrophage, stroma, brain, liver, muscle, andtestes.

Example 3 Purification of 312C2 Protein

Multiple transfected cell lines are screened for one which expresses theantigen at a high level compared with other cells. Various cell linesare screened and selected for their favorable properties in handling.Natural 312C2 can be isolated from natural sources, or by expressionfrom a transformed cell using an appropriate expression vector.Purification of the expressed protein is achieved by standardprocedures, or may be combined with engineered means for effectivepurification at high efficiency from cell lysates or supernatants. FLAGor His₆ segments can be used for such purification features.

By Northern analysis, it is clear that 312C2 is expressed in variousTh1, Th2, CD4+, CD8+, NK1.1+, pro-, pre-, and αβCD4−CD8− T cells. 312C2is also expressed in thymus and activated spleen cells. Cells expressing312C2 typically contain a transcript of about 1.3 kb, corresponding tothe size of the cloned 312C2 cDNA. Transcripts for 312C2 have not beendetected in heart, kidney, macrophage, stroma, brain, liver, muscle,testes tissue.

The structural homology of 312C2 to the TNF receptor family, suggests abroad function of this molecule. 312C2, as an activation molecule,likely mediates enhanced Ag-specific proliferative responses on T cells,or induction of apoptosis of these cells. 312C2 agonists, orantagonists, may also act as a co-stimulatory molecule for T-cellactivation, and may in fact, cause a shift of T helper cell types, e.g.,from Th1 to Th2, or Th2 to Th1. Thus, 312C2 may be useful in thetreatment of abnormal immune disorders, e.g., T cell immunedeficiencies, chronic inflammation, or tissue rejection.

The mouse 312C12 protein exhibits structural features characteristic ofa cell surface antigen. The protein is easily detected on particularcell types, others express lesser amounts. The 312C2 antigen should bepresent in the identified tissue types and the interaction of theantigen with its binding partner should be important for mediatingvarious aspects of cellular physiology or development.

Example 4 Isolation of Homologous 312C2 Genes

The 312C2 cDNA can be used as a hybridization probe to screen a libraryfrom a desired source, e.g., a primate cell cDNA library. Many differentspecies can be screened both for stringency necessary for easyhybridization, and for presence using a probe. Appropriate hybridizationconditions will be used to select for clones exhibiting specificity ofcross hybridization. Specifically, the mouse 312C2 cDNA clone was usedto probe the HY06 human anergic T cell library. A clone of about 1006 bpencoding a predicted polypeptide of 241 amino acids was isolated.

Screening by hybridization using degenerate probes based upon thepeptide sequences will also allow isolation of appropriate clones.Alternatively, use of appropriate primers for PCR screening will yieldenrichment of appropriate nucleic acid clones. See SEQ ID NO: 5.

Similar methods are applicable to isolate either species, polymorphic,or allelic variants. Species variants are isolated using cross-specieshybridization techniques based upon isolation of a full length isolateor fragment from one species as a probe.

Alternatively, antibodies raised against mouse 312C2 will be used toscreen for cells which express cross-reactive proteins from anappropriate, e.g., cDNA library. The purified protein or definedpeptides are useful for generating antibodies by standard methods, asdescribed above. Synthetic peptides or purified protein are presented toan immune system to generate monoclonal or polyclonal antibodies. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; andHarlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press. The resulting antibodies are used for screening, panning,or sorting.

Example 5 Expression and Tissue Distribution of Human 312C2

Southern and PCR analysis of various hematopoietic cells and tissues wasperformed as described above. Expression was detected in several celllines and tissues, most notably, stimulated dendritic cell library, someactivated T cell clones, activated PBMCs, NK clones, Th1, Th2 cells,pre-T cells, pro-T cells. Spleen and lung tissue had detectable levelsof 312C2.

Example 6 Preparation of Antibodies Specific for 312C2

Synthetic peptides or purified protein are presented to an immune systemto generate monoclonal or polyclonal antibodies. See, e.g., Coligan(1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press.Polyclonal serum, or hybridomas may be prepared. In appropriatesituations, the binding reagent is either labeled as described above,e.g., fluorescence or otherwise, or immobilized to a substrate forpanning methods.

Example 7 Chromosomal Mapping of 312C2

Chromosome spreads are prepared. In situ hybridization is performed onchromosome preparations obtained from phytohaemagglutinin-stimulatedlymphocytes cultured for 72 h. 5-bromodeoxyuridine is added for thefinal seven hours of culture (60 μg/ml of medium), to ensure aposthybridization chromosomal banding of good quality.

An appropriate fragment, e.g., a PCR fragment, amplified with the helpof primers on total B cell cDNA template, is cloned into an appropriatevector. The vector is labeled by nick-translation with ³H. Theradiolabeled probe is hybridized to metaphase spreads as described inMattei, et al. (1985) Hum. Genet. 69:327-331.

After coating with nuclear track emulsion (KODAK NTB₂), slides areexposed, e.g., for 18 days at 4° C. To avoid any slipping of silvergrains during the banding procedure, chromosome spreads are firststained with buffered Giemsa solution and metaphase photographed.R-banding is then performed by the fluorochrome-photolysis-Giemsa (FPG)method and metaphases rephotographed before analysis.

Example 8 Isolation of Variants from Individuals

Mutational 312C2 variants from individuals having abnormal immuneresponses are isolated by standard methods. For example, affected cells,e.g., lymphocytes, are isolated as described, e.g., in Coligan (1991)Current Protocols in Immunology Wiley/Greene, NY. cDNA libraries areconstructed as described above and probed with the mouse or human 312C2clone. Isolated clones are then sequenced and compared to the human ormouse clone.

Alternatively, PCR techniques are also be employed to isolate variants.See, e.g., Innis, et al. (eds.)(1990) PCR Protocols: A Guide to Methodsand Applications, Academic Press, N.Y.

Example 9 Immunohistochemical Localization

The antibody described in Example 6 is used to identify expression of312C2 in various tissues. Methods for immunohistochemical staining aredescribed, e.g., in Sheehan, et al. (eds.) (1987) Theory and Practice ofHistotechnology, Battelle Press, Columbus, Ohio

Example 10 Soluble Molecules

Soluble constructs of 312C2 are made, e.g., as described in Ausubel, etal. (1987 and Supplements) Current Protocols in Molecular Biology,Greene and Wiley, NY; and Coligan, et al. (eds.) (1995 and periodicsupplements) Current Protocols in Protein Science, John Wiley and Sons,NY. Briefly, the transmembrane portion of 312C2 is truncated. Thenucleic acid encoding the remaining portions of the molecule issubcloned into an appropriate vector and the protein expressed through asuitable host cell.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described above are offeredby way of example only, and the invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. All references cited herein areincorporated herein by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

What is claimed is:
 1. A substantially pure or recombinant 312C2 proteinor conservatively modified variants thereof, said protein: a) isexpressed on activated T cells; or b) specifically binds to antibodiesgenerated against SEQ ID NO: 2 or
 4. 2. A protein or peptide of claim 1,selected from the group consisting of: a) a natural protein from a warmblooded animal selected from the group of birds and mammals, including arodent or primate; b) a protein or peptide: i) comprising at least onepolypeptide segment of at least 14 amino acids of SEQ ID NO: 2; ii)comprising at least one polypeptide segment of at least 14 amino acidsof SEQ ID NO: 4; or iii) which is not glycosylated; iv) which is in abuffered solution; v) which is attached to a solid substrate; vi) whichexhibits a plurality of epitopes from SEQ ID NO: 2 or 4; vii) which isdetectably labeled; viii) which is a synthetic polypeptide; ix) which isconjugated to a chemical moiety; x) which is a 5-fold or lesssubstitution from a natural sequence; or xi) which is a deletion orinsertion variant from a natural sequence.
 3. A protein or peptide ofclaim 1, comprising a sequence from the extracellular or theintracellular portion of a 312C2.
 4. A fusion protein comprising apeptide of claim
 1. 5. A sterile composition comprising the protein ofclaim 1, and a pharmaceutically acceptable carrier.
 6. An antibody whichspecifically binds a protein or peptide of claim
 1. 7. An antibody ofclaim 6, wherein: a) said 312C2 is a mammalian protein, including amouse or human; b) said antibody is raised against a purified peptidesequence of SEQ ID NO: 2 or 4; c) said antibody is a monoclonalantibody; d) said antibody is detectably labeled; e) said antibody isattached to a solid substrate; f) said antibody is in a sterilecomposition; or g) said antibody is in a buffered composition.
 8. Amethod of purifying a 312C2 protein or peptide from other materials in amixture comprising contacting said mixture to an antibody of claim 6,and separating bound 312C2 from other materials.
 9. An isolated orrecombinant nucleic acid capable of encoding a protein or peptide ofclaim
 1. 10. A nucleic acid of claim 9, wherein said nucleic acid: a)encodes a sequence of SEQ ID NO: 2 or 4; b) comprises a sequence of SEQID NO: 1 or 3; or c) encodes a sequence from an extracellular domain ofa natural 312C2; d) encodes a sequence from an intracellular domain of anatural 312C2; e) attached to a solid substrate; f) is detectablelabeled; or g) is in a sterile composition.
 11. An expression orreplicating vector of claim
 9. 12. A kit comprising: a) a substantiallypure 312C2 or fragment of claim 1; b) an antibody or receptor whichspecifically binds a 312C2; or c) a nucleic acid encoding a 312C2 orpeptide.
 13. A method for detecting in a sample the presence of a 312C2nucleic acid, protein, or antibody, comprising testing said sample witha kit of claim
 12. 14. A method of modulating the physiology of a cellcomprising contacting said cell with: a) a substantially pure 312C2 orfragment of claim 1; b) an antibody or binding partner whichspecifically binds a 312C2; or c) a nucleic acid encoding a 312C2 orpeptide.
 15. The method of claim 14, wherein said cell is a T cell andsaid modulating of physiology is: a) apoptosis of said T cell; or b)activation of said T cell
 16. A method of claim 14, wherein said cell isin a tissue and/or in an organism.
 17. A method of expressing a 312C2comprising expressing a nucleic acid of claim
 9. 18. A cell, tissue,organ, or organism comprising a nucleic acid of claim
 9. 19. Arecombinant nucleic acid comprising sequence at least about 70% identityover a stretch of at least about 30 nucleotides to a 312C2 nucleic acidsequence of SEQ ID NO: 1 or
 3. 20. A nucleic acid of claim 19, furtherencoding a polypeptide comprising at least about 60% identity over astretch of at least about 20 amino acids to a 312C2 sequence of SEQ IDNO: 2 or
 4. 21. A method of treating a host mammal having an abnormalimmune response by administering to said mammal an effective dose of: a)an antibody or binding partner which binds specifically to a 312C2; b) asubstantially pure 312C2 protein or peptide thereof; or c) a nucleicacid encoding a 312C2 peptide.
 22. The method of claim 21, wherein saidabnormal immune response is characterized by: a) a T-cell immunedeficiency; b) chronic inflammation; or c) tissue rejection.